okadaic-acid and phosphohistidine

Kinetic constants of KM = 0.8 microM, 3 microM and 1.6 microM, and kcat = 9 s-1, 7 s-1 or 9 s-1 were determined for histidine dephosphorylation by protein phosphatases 1, 2A and 2C respectively. IC50 values were determined for the inhibition of protein phosphatase 1 by inhibitor 1 (IC50 = 1 nM), inhibitor-2 (IC50 = 3 nM) and okadaic acid (IC50 = 30 nM) and for the inhibition of protein phosphatase 2A by okadaic acid (IC50 = 0.02 nM) and microcystin-LR (IC50 = 1 nM). Inhibitor-1 (Ki = 0.7 nM) and okadaic acid (Ki = 32 nM) are noncompetitive with protein phosphatase 1. Some of the IC50 values were low enough to violate the assumptions of the usual inhibition equations and a more general approach to the analysis of the data was used. On the basis of these kinetic parameters and the presence of phosphohistidine, the major cellular protein serine/threonine phosphatases are likely to act as protein histidine phosphatases in the cell.

Topics: Animals; Ethers, Cyclic; Histidine; Kinetics; Marine Toxins; Microcystins; Okadaic Acid; Peptides, Cyclic; Phosphates; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Phosphatase 2; Rabbits

Eukaryotic cellular proteins contain phosphohistidine. To search for protein histidine phosphatases, protein histidine kinase from Saccharomyces cerevisiae was used to phosphorylate histone H4 on histidine at position 75 in the H4 amino acid sequence. Incubation of the phosphorylated histone H4 with either protein phosphatase 1, 2A, or 2C resulted in extensive removal of phosphate from the phosphorylated histone. Thus, protein phosphatases 1, 2A, and 2C are histidine phosphatases as well as serine/threonine phosphatases. Calcium/calmodulin-regulated protein phosphatase (protein phosphatase 2B) did not remove phosphate from phosphohistidine. The histidine phosphatase reaction was tested for a magnesium requirement and effects of inhibitor-1 and okadaic acid. In all cases, the protein phosphatases behaved as they do in their serine/threonine phosphatase activity. Extracts of the yeast, S. cerevisiae, contain protein histidine phosphatase activity. Quantitative measurement of phosphatase activity shows that the activity against phosphohistidine is a major activity of protein phosphatases 1, 2A, and 2C.

Topics: Carrier Proteins; Ethers, Cyclic; Histidine; Histidine Kinase; Histones; Intracellular Signaling Peptides and Proteins; Kinetics; Magnesium; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Phosphatase 1; Proteins; Saccharomyces cerevisiae; Ultrafiltration